The mucosal surfaces in animals and humans, especially the gastrointestinal (GI) and respiratory tracts, are the major portals of entry and/or sites of diseases caused by bacterial, viral, and parasitic pathogens. Examples of these diseases include those caused by enteropathogenic Escherichia coli, Campylobacter sp., Salmonella sp., Listeria monocytogenes, Helicobacter pylori, Shigella sp., rotaviruses and calciviruses in the gastrointestinal (GI) tract, and Mycoplasma pneumoniae, influenza virus, Mycobacterium tuberculosis, Streptococcus pneumoniae, severe acute respiratory syndrome (SARS) virus and respiratory syncytial virus in the respiratory tract. The urogenital tract is also a site of mucosal invasion/disease (e.g., those caused by human immunodeficiency virus, Neisseria and Chlamydia). The mucosal surfaces, especially in the respiratory tract, are also the sites where allergens (for example dust mites, pollen etc.) cause hyper immune responses resulting in allergic airway diseases such as asthma.
However, the majority of current, licensed, human and veterinary vaccines are administered parenterally or systemically (for example by subcutaneous, intramuscular, intraperitoneal routes), and although these elicit immunity in the systemic compartment (bone marrow, spleen and lymph nodes), they fail to elicit immunity in the functionally independent mucosal compartment (lymphoid tissues in the mucosae and external secretory glands). For example, the inactivated polio virus vaccine administered systemically may prevent the development of poliomyelitis, but it fails to prevent infection in the GI tract or in the tonsils.
Systemic immunization fails to induce mucosal IgA (Singh and O'Hagan, 2002). It is also acknowledged that vaccines that promote strong immune responses when given via the systemic route, may not be as successful if administered via the mucosal route (Neutra and Kozlowski, 2006). Conversely, mucosally administered vaccines that elicit mucosal immunity do not necessarily elicit systemic immunity also (Carol and Nieto, 1998).
Development of safe vaccines that elicit strong and prolonged mucosal immunity that would circumvent the attachment/colonization of pathogens to the mucosal epithelium, impede pathogen replication/penetration in the mucosa, and/or block activity of microbial toxins, would be a significant advancement in the prevention and treatment of a wide spectrum of infectious diseases. Also, mucosal immunity would be beneficial for elderly people since it is not subject to age-associated dysfunction, as well as for very young people since mucosal immunity appears to develop earlier than the systemic immunity (O'Hagan, 1998). This is a current, major, global aim in the field of vaccinology.
Furthermore, mucosal immunization can also be used for inducing immunological tolerance (immunologic hyporesponsiveness) and can serve as an attractive strategy for preventing or treating disorders resulting from untoward inflammatory immune reactions against self—(i.e., auto) or non-self-antigens, such as in type 1 diabetes, asthma, multiple sclerosis, autoimmune uveitis, and rheumatoid arthritis (see Ogra et al., 2001). Mucosal administration of relevant autoantigens and allergens on their own (in the absence of other known immune modulating components), has been shown to delay or suppress onset of clinical disease in a number of experimental autoimmune and allergic disorders. However, this approach often requires repeated feeding of large amounts of antigens over long periods and is only partly effective in the treatment of already established diseases. Mucosal administration of appropriately adjuvanted autoantigens has the potential to effectively suppress systemic auto-T cell reactivity and induce the selective migration and retention of protective/regulatory T cells into lymphoid tissues and organs involved.
It is understood that CD8+ cytotoxic T-lymphocyte (CTL) responses involving participation of CD8+ T cells are essential for host protection against intracellular pathogens since these cells are capable of killing the infected host cells. CTL responses are also known to be required for the killing of neoplastic host cells (Henkart, 1997; Singh and O'Hagan, 2002). Compared to the oral (per oral, p.o.) route, the intranasal (i.n.) route is generally more favourable and efficacious, and requires less dose of the immunogen since it does not have to encounter the relatively harsher environment of the gastrointestinal route (Davis, 2001; Lemoine et al., 1998; Singh and O'Hagan 2002).
Immune mechanism(s) to control infectious diseases requires the induction of neutralizing antibodies (humoral immunity) and generation of cell mediated immunity (CMI), including CD4+ helper T (Th) cells, and CD8+ cytotoxic (cytolytic) T lymphocytes (CTL). Naïve CD8+ T cells are stimulated when peptides from endogenously derived antigens are presented in the context of MHC class I molecules. Although this process can occur virtually in all cells, only self-peptides, or peptides derived from viral or bacterial proteins being assembled within the host cell, are presented on MHC class I. Upon activation, naïve CD8+ T cells differentiate into effectors and memory T cells that possess the ability to kill infected target or tumor cells.
On the other hand, protein antigens from the extracellular fluids that are taken up by antigen presenting cells (APCs) through pinocytosis, or phagocytosis in the case of particulate antigens, are fragmented within endosomes. The peptides generated are presented by the APCs in the context of MHC class II molecules and stimulate CD4+ T cells. CD4+ T helper cells contribute towards control of infection primarily by producing cytokines that aid antibody responses, inflammation, macrophage activation, and CD8+ T cell proliferation (Krishnan and Mosmann, 1998).
The mammalian mucosal immune system comprises innate, non-specific defenses, and the adaptive immunologic network consisting of the gut-associated lymphoid tissue (GALT), bronchoepithelium and lower respiratory tract (BALT), ocular tissue, upper airway, tonsils, salivary glands and nasopharynx (NALT), and the larynx (LALT). There is some commonality in the mucosal immune network which has been referred to as the common mucosal immune system, and immunization at one mucosal surface has been shown to elicit immunity at a distal mucosal site. M-cells present in the epithelium of inductive mucosal sites (these sites are replete with B cells, T cells, dendritic cells, and also macrophages) are important in the transport of luminal antigens. Peyer's patches (PP) and NALT are the most commonly identified repositories of the M-cells. Secretory IgA (S-IgA) is the best-defined humoral effector component of the mucosal immune system; it is associated with M-cell-mediated antigen uptake. However, T lymphocytes of the CD4+ and CD8+ phenotypes and not IgA B cells, are the major effector cells present in the mucosal surfaces (Ogra et al., 2001).
Successful vaccination typically depends on two main criteria: identification of relevant antigenic target(s) for the pathogen or the disease, and the ability to evoke an appropriate protective or beneficial immune response, against the pathogen or the disease, in the vaccinated host. Some of the current research approaches for developing mucosal vaccines are based on the use of live-attenuated pathogens, or killed whole pathogen cells or components thereof, or the use of live viral/bacterial vectors (e.g. vaccinia, poxviruses, adenoviruses; Salmonella, BCG, Bordetella, commensal bacteria such as lactobacilli). Research approaches for non-replicating mucosal delivery vehicles and adjuvants have included heat shock proteins and microparticulates such as virosomes (reconstituted lipid vesicles consisting of viral glycoproteins), liposomes (closed lipidic vesicles made from ester lipids), cochleates (ester lipid liposomes converted into rolled up structures devoid of aqueous compartments by treatment with cations), polymeric microspheres, mucoadhesive polymers, ISCOMS (cage-like complexes consisting of glycosides of Quil A as the adjuvant in conjunction with phospholipids and cholesterol), virus like particles, CpG oligonucleotides and DNA as delivery/adjuvant systems, and the use of bacterial toxins such as cholera toxin (CT) and heat labile toxin (LT) from E. coli as potent mucosal adjuvants (Holmgren et al., 2003; Kemble and Greenberg, 2003; Lemoine et al., 1998; Ogra et al., 2001).
However, all of these approaches have drawbacks related to either safety, ease of preparation and/or efficacy. There are regulatory concerns regarding the use of ill-defined whole cell vaccines. The live attenuated pathogens may revert to virulence, especially posing a risk for the immuno-compromised population. There is potential for genetic integration between the attenuated and wild type strain that could result in creation of a more virulent strain. There maybe stronger immunity generated against the bacterial/viral vector than the antigen of interest, pre-existing immunity against the vector may reduce the efficacy of the vaccine in such pre-disposed population. There remain serious public/regulatory concerns regarding CpG and DNA vaccines (potential integration with the host DNA, tissue damage by CpG) in addition to issues concerning sustenance and duration of immunity generated. CT and LT holotoxins are highly toxic to most animals and humans, and their less toxic variants have diminished efficacies (Holmgren et al., 2003; Lemoine et al., 1998; Mestecky et al., 1997; Ogra et al., 2001). Some vaccines also require very complex methods for formulating. As a result, there are a few mucosal vaccines currently on the market, all using live-attenuated or dead whole cells.
Considerable progress has been made over the past decade using modern molecular biology and genomics approaches, towards the identification, purification and/or synthesis of key antigenic determinants of pathogens and non-pathogenic diseases such as tumors and allergies. However, such highly purified antigens (proteins and/or peptides, carbohydrates etc.) are relatively weak immunogens, limiting their ability to induce strong protective immune responses. Vaccines using acellular (non-replicating) subunit (purified or highly purified), antigens are preferred from safety and regulatory perspectives.
The poor immune responses of non-replicating antigen vaccines given mucosally is at least partially due to the lack of transport of the antigen through the epithelial layer to the mucosal immune network, and the rapid deactivation/elimination of the antigen (Jakobsen and Jonsdottir, 2003). This has continued to intensify global research aimed at developing safe and effective mucosal adjuvants, and effective mucosal vaccine delivery systems for elicitation of protective immunity. Some researchers have resorted to evaluating the use systemic prime/mucosal boost immunization strategy in order to elicit a stronger mucosal immune response (Lauterslager et al., 2003; O'Hagan, 1998). However, although there are many experimental mucosal adjuvants and mucosal delivery systems, none of these has been approved for commercial use to-date.
An adjuvant is recognized in the vaccine art as a substance or material which when administered together or in conjunction with an immunogen (antigen) increases the amount and quality of the immune response to that immunogen in the vaccinated/immunized host. Adjuvants are used in vaccine compositions to elicit an immune response sooner, or a greater response, or with less immunogen, or to increase or to suppress, production of certain antibody subclasses (potentiate the type of immune response desired for specific application) that afford immunological protection or to provide memory response to the immunogen, or to enhance/sustain components of the immune response.
Adjuvants may be further demarcated as being systemic and mucosal, based on the different physiological conditions of antigen delivery and processing that result in the generation of distinct immune responses. Mucosal vaccine adjuvants are those that are effective in eliciting of mucosal immunity, or systemic immunity, or mucosal as well as systemic immunity to an antigen in a vaccine that is administered via the mucosal route. Mucosal adjuvants can be broadly classified as those that play an immunostimulatory role (for example toxin-based, cytokine-based, innate immunity associated) and those that facilitate vaccine or antigen delivery (for example poly D,L-lactide-co-glycolide or PLGA, liposomes, cochleates, live-attenuated vectors, chitans, mucoadhesives, DNA vaccines) for the induction of a protective immunity (Holmgren at al., 2003; Ogra et al., 2001; Yuki and Kiyono, 2003).
The adjuvanticity (adjuvant activity) of mucosal adjuvants is partly manifested by their ability to help the antigen traverse the mucosal barrier imposed by the route of administration. Once the antigen has been assimilated, the adjuvant may impact the immunity by any of the recognized means in the art, such as complement activation, antigen adsorption and depot effect, induction of cytokines, presentation of the antigen to various antigen presenting cells (APCs), regulation of the expression through MHC class I or class II, stimulation of antibody production or antibody type switching (McElrath, 1995).
The bacterial protein enterotoxins of Vibrio cholerae (CT, cholera toxin) and of enterotoxicogenic E. coli (LT, heat labile enterotoxin) have been the most widely studied for development of mucosal vaccines. Although they are effective in eliciting of mucosal (e.g., IgA) as well as systemic immune responses (e.g. serum IgG), these holotoxins are too toxic for human/veterinary applications. On the other hand, their less toxic derivatives are not as effective (Arakawa et al., 1998; Holmgren et al., 2003; Ogra et al., 2001; Yuki and Kiyono, 2003).
Alum (aluminium hydroxide) is the only adjuvant currently approved universally for use in humans. However, it fails to elicit mucosal IgA antibodies upon p.o. or i.n. immunization (Alpar et al., 1992; Singh and O'Hagan, 2002). Thus, adjuvants that work for eliciting of systemic immunity may not be predictive for the elicitation of mucosal immunity.
For particulate or microparticulate mucosal delivery vehicles/carriers and adjuvants, it is recognized that the size of the particles has an impact on the type of immune response elicited. However, there is no clear consensus on the size ranges for optimal efficacy (Ogra et al., 2001; O'Hagan, 1998; Rubido et al., 2002).
Liposomes are closed, lipid vesicles containing an entrapped (encapsulated) aqueous volume. The hydrophilic polar head groups of the lipids forming liposomes are oriented towards the aqueous environment present inside and outside the liposome, whereas the hydrophobic “tail” region of the lipid is sandwiched between the polar head groups and away from the aqueous environment. Liposomes can vary in size from <50 nm to several micrometers in diameter, depending on the lipids used and the method of preparation. Depending on the methods used for making them, the liposomes can be unilamellar (one bilayer) or multilamellar (several bilayers with aqueous compartments between the adjacent bilayers).
Liposomes are well recognized in the art. Methods to encapsulate materials within the aqueous compartments of liposomes, and/or to associate with the hydrophobic lipid layer, are well known to those skilled in the art. These methods are exemplified by, but not limited to, detergent dialysis, dehydration-rehydration, reverse-phase evaporation, sonication, pressure extrusion, and remote loading. Liposomes composed predominantly of ester lipids, with or without additional components such as a sterol, are referred to herein as conventional liposomes or liposomes.
Liposomes on their own are not effective mucosal adjuvants, requiring the presence/association of additional known adjuvants or targeting molecules such cholera toxin B subunit, CT, dimethyl dioctadecyl ammonium bromide, or acemannan polysaccharide (Baca-Estrada et al., 2000; Harokopakis et al., 1998; Mestecky et al., 1997; Rubido et al., 2002; Vadolas et al., 1995), or they require high doses (1.6 mg/8-10 week old Balb/c mouse) of the liposomal lipid (de Haan et al., 1995) for efficacy. The addition of targeting molecules, on the surfaces of liposomes, specific to the mucosal epithelium to enhance the efficacy of mucosal delivery has also been suggested to improve the efficacy of liposomes as mucosal adjuvants (Mestecky et al., 1997; Ryan et al., 2001; Yuki and Kiyono, 2003).
Cochleate cylinders, formed via cation-induced (for example, Ca2+ or Mg2+) fusion of conventional phosphatidyl serine containing liposomes, were first described by Papahadjopoulos and colleagues, who used these as intermediate structures for conversion into large unilamellar liposomes by subsequent chelation of the added cations with EDTA (Papahadjopoulos, 1978; Papahadjopoulos et al., 1975). Later, others described various different methods for the interaction of cations for the conversion of conventional liposomes into cochleate structures, together with the incorporation/association of different molecules, and the application of the cochleates for the delivery of the said different molecules via various routes of administration. These disclosures are expressly incorporated herein by reference (Gould-Fogerite and Mannino, 1996, 1997, 1999; Gould-Fogerite et al., 1998; Mannino and Gould-Fogerite, 1998; Jin, 2004; Jin et al. 2000; Margolis et al., 2002; Zarif et al., 2003; Zarif and Tan, 2003).
These references are in agreement that the interaction of cations with negatively charged lipids, comprising liposomes made from conventional ester lipids, results in the conversion of the liposomes into cochleate cylinder structures. The cochleate structures consist of various sizes of continuous, solid, lipid bilayer sheets rolled up in a spiral (like a jelly roll). These structures are devoid of any internal aqueous spaces, and they appear as “needle like” structures under microscopic examination. These structures have also been referred to as nonaqueous structures. The average diameters of the cochleate cylinders can range from 40 nm to several μm (Jin, 2004; Mannino and Gould-Fogerite, 1998).
As such, cochleates are distinct from liposomes which are individual, spherical, closed vesicles which essentially do contain encapsulated internal aqueous compartment(s). In liposomes, water soluble molecules are encapsulated within the aqueous compartment(s) and hydrophobic molecules can be associated with the hydrophobic tail region of the lipid bilayer where it can be entirely within the hydrophobic region and/or could be exposed on the outer surface of the vesicle or oriented inside towards the encapsulated aqueous compartment. In cochleates, the molecules of interest for delivery purposes are entrapped within the rolled up lipid bilayer sheets.
It is recognized that the lipids used for making the liposomes need to have a net negative charge for the subsequent interaction with the added cations for conversion to cochleates. In addition, although lipids that are negatively charged by the presence of phosphatidyl serine head groups are amenable for cochleate formation, not all negatively charged lipids/liposomes could be converted into cochleates (Papahadjopoulos, 1978; Gould-Fogerite et al., 1998). Moreover, some lipids needed to be purified to at least 75% enrichment of phosphatidyl serine (by weight), in order for being able to convert the liposomes made therefrom into cochleates cylinders (Zarif and Tan, 2003). For preparation of cochleates from ester lipids (which in many instances contain some degree of unsaturation in their fatty acyl chains), the cochleate manufacturing process is conducted under an inert gas such as nitrogen, and the cochleates are stored under an inert gas (Jin, 2004; Jin et al., 2000; Zarif et al., 2003) to avoid potential stability- and toxicity-related problems associated with lipid oxidation.
The conversion of conventional liposomes to cochleates is mediated by cation-induced fusion of the vesicles (Jin, 2004; Margolis et al., 2002; Papahadjopoulos 1978). It has been reported that the conversion of conventional liposomes into cochleate structures by addition of divalent cations such as Ca2+ required incubation periods of from one to several hours (Papahadjopoulos, 1978; Papahadjopoulos et al., 1977). For making cochleates from conventional liposomes, a molar concentration of Ca2+ that is up to one half the molar concentration of the lipids/phospholipids (that is, the molar ratio of lipids to cations of 1:0.5) is required (Gould-Fogerite et al., 1998; Margolis et al., 2002).
More recently, cationic organic drugs have been used to directly convert conventional liposomes into cochleates and entrap the drug, rather than using cations to form cochleates from liposomes (Jin, 2004). Several methods have been described in the literature, for preparation of cochleate formulations, incorporating different means of adding cations to liposomes to convert them into cochleates (Jin, 2004; Jin et al., 2000; Papahadjopoulos, 1978; Papahadjopoulos et al., 1975; Zarif et al., 2001, 2003; Gould-Fogerite, S., and Mannino, 2000). These methods have been referred to variously as standard cochleates (liposomes are pre-formed by hydration/sonication of lipids followed by either direct addition of calcium or infusion of calcium via dialysis against a calcium containing buffer), DC cochleates (infusion of calcium at the same time as liposomes are being formed by detergent dialysis process), LC cochleates (liposomes made by detergent dialysis followed by calcium addition separately), hydrogel-isolated cochleate (liposomes with the loaded components are added to a polymer A which is then mixed with another polymer B in which polymer A is not miscible, resulting in an aqueous two-phase system to which is then added a cation salt so that it diffuses into the polymer A containing liposomes.
In all these methods, except the one where the molecule of interest is a cationic molecule added directly to empty pre-formed liposomes to make cochleates (Jin, 2004), the molecule of interest is first encapsulated/associated with the liposomes, and the cation is added without prior removal of the un-encapsulated (or material not associated with the liposomes) molecules present in the external milieu, raising the strong possibility that there could be substantial amounts of the molecule present in the formulation as a cation-precipitate, besides that entrapped in the cochleate structures. This external precipitated material is mostly left with the cochleate formulation since there is no simple way to remove this from the rest of the cochleate which also is a precipitate. In addition, the presence of this material in the formulation could have a significant impact on the observed biological effects of molecule delivered using the so called cochleate formulation. One skilled in the art will recognize that there are various means such as for example ultracentrifugation or dialysis using an appropriate molecular weight cut off, to allow escape of the un-encapsulated molecules from the liposome preparation, before addition of the cation to make cochleates, so as to avoid the possibility of cation-precipitated molecule being present in the formulation. One skilled in the art will also recognize that there could be many other means for addition of cations to liposomes to convert them into cochleate structures. One skilled in the art will also appreciate that any molecule of interest, whether it is hydrophilic or hydrophobic in characteristic, can be entrapped/associated in/with cochleate structures.
Lipids amenable for cochleate formation can be obtained from animal or plant membranes (Mannino and Gould-Fogerite, 1998), but there is no suggestion or mention that lipids obtained from the membranes of microorganisms, in the absence of other bacterial components such as membrane proteins and lipopolysaccharides (LPS), would be amenable for cochleate formation. Cochleates have recently been made from lipid-containing outer membrane extracts of Neisseria meningitides, but these extracts also contained major outer membrane proteins and LPS (Bracho et al., 2006; Campo et al., 2006; Perez et al., 2006). The advantages of cochleates as delivery vehicles, compared to the liposomes from which they have been converted from, have been attributed to improved stability properties of these structures, the protection of the associated biological molecules from the external environment, and the ability to store lyophilized cochleate preparations that can be re-hydrated just prior to use without loss of the associated biological molecules (Mannino and Gould-Fogerite, 1998; Margolis et al., 2002; Zarif et al., 2003).
Cochleates with associated drugs and other bioactive molecules, including antigens, have been shown to be useful for the delivery of these compounds in murine models (Gould-Fogerite et al., 1998; Gould-Fogerite and Mannino, 1999; Jin et al., 2000; Margolis et al., 2002; Zarif et al., 2003). The use of cochleates for delivery of antigens in vaccine applications has been described for vaccines administered by parenteral (such as i.m.), per oral (p.o.) or a combination of both routes (Gould-Fogerite and Mannino, 1996; Gould-Fogerite et al., 1998; Gould-Fogerite et al., 1997; Jin et al., 2000).
Cochleate/protein vaccines (synthetic lipid dioleyl phosphatidylserine having 18-carbon acyl chains and one unsaturated bond was used as the negatively charged lipid for making the formulation) administered p.o. or i.m. were stated to elicit strong circulating mucosal antibodies, but no data were given to illustrate this, to be able to discern the level of immune response (Gould-Fogerite and Mannino, 1996). The paper also stated that i.n. administration elicited circulating antibodies but did not specify what subtype the antibodies were, and did not mention or suggest that any of the antibodies were of the IgA isotype indicative of a mucosal immune response.
It has been previously reported that i.n. immunization with vaccine wherein the antigen is associated with polymeric particles, may elicit an increase in the circulating antibodies of the IgG isotype but not of the IgA isotype (Lemoine et al., 1998). This shows that the mere mentioning of elicitation of circulating antibodies without identifying the specific mucosal suggestive antibody subtype is not indicative of the elicitation of a mucosal immunity.
Administration of protein- or glycoprotein/cochleates vaccines via p.o. or i.m. routes were reported to elicit specific serum IgA responses, especially after the second immunization (Gould-Fogerite et al., 1998). In most other experimental vaccines, systemic immunization fails to elicit an IgA response (Singh and O'Hagan, 2002), and systemic immunization fails to generate protective immunity at the mucosal surfaces (Yuki and Kiyono, 2003).
The p.o. and i.m. immunization with PS-cochleate/glycoprotein vaccine elicited antigen specific serum IgG1 and IgG2a responses also, as did the corresponding PS-liposome vaccine (Gould-Fogerite et al., 1998). This paper also reported that the serum IgA antibody response upon p.o. immunization with PS-liposomes containing glycoproteins was similar or greater than that obtained with corresponding PS-cochleates-glycoproteins.
It was reported that the immune responses obtained with the cochleate vaccine formulations administered p.o. or i.m. were slower to develop than those obtained with liposomes, but the cochleate vaccine responses kept on steadily increasing for a period of several months after immunization (Gould-Fogerite et al., 1998; Gould-Fogerite and Mannino, 1996). Although immune response data are given for cochleate vaccines administered via the p.o., i.m. or a combination of both routes, there was no indication or suggestion of the elicitation of a mucosal immune response upon i.n. administration (Gould-Fogerite and Mannino, 1997). There are differences in the adsorption of the antigen administered via the p.o. and the nasopharyngeal routes such as i.n. In addition, an adjuvant effective via the p.o. route is not predictive of its efficacy by the nasopharyngeal route (Rubido et al., 2002).
Elicitation of anti-OVA IgA responses in saliva, but not serum IgG responses, were recently reported upon i.n. immunization of mice with OVA formulated in cochleates made from proteoliposomes consisting of outer membrane proteins, lipids and LPS extracted from N. meningitidis (Bracho et al., 2006). However, it has been reported that mucosal IgA and systemic IgG immune responses are elicited against the proteoliposome components of these cochleates (Campo et al., 2006; Perez et al., 2006), raising concerns about the efficacy of such cochleates for use in subsequent immunizations due to the pre-existing immunity against the carrier. Also, it should be noted that the LPS (an endotoxin) in these cochleates is a known immunostimulating molecule.
Archaea is a Domain of microorganisms that is considered to be distinct from the two Domains constituting eubacteria and eukaryotes. Archaea includes aerobic, anaerobic (including methanogenic), thermophilic, extremely thermophilic, thermoacidophilic, and extremely halophilic microorganisms. The unique polar lipid structures of archaeal membranes are one of the key, unique defining characteristic that helps distinguish members of Archaea from species of the other two domains. The total lipids extracted from archaeal species consist of between 80-95% polar lipids, the balance being neutral lipids.
Archaeal polar lipids are composed of branched phytanyl chains, usually of constant length, which are fully saturated in most species. They are uniquely attached via ether bonds to the glycerol backbone carbons at the sn-2,3 positions (Kates, 1992). In contrast, conventional ester phospholipids found in members of the domains Bacteria and Eukarya have fatty acyl chains of variable length, which may be unsaturated, and these are distinctly attached via ester bonds to the sn-1,2 carbons of the glycerol.
The core structures of archaeal polar lipids (the polar head groups having been removed by hydrolysis) consist of the standard archaeol (also referred as diether) lipids (2,3-di-O-phytanyl-sn-glycerol) and/or the standard caldarchaeol (also referred as tetraether) lipids (2,2′,3,3′-tetra-O-dibiphytanyl-sn-diglycerol) and modifications thereof (Kates, 1992). Diether lipids are monopolar like the conventional ester phospholipids, whereas the tetraether lipids are bipolar. The polar head groups, attached to the sn-1 glycerol carbon in the archaeols and to the sn-1 and sn-1′ glycerol carbons in the caldarchaeols, can vary and may include phospho groups, glyco groups, phosphoglyco groups, polyol groups, or hydroxy groups.
The phosphatidylcholine head group commonly encountered in conventional ester lipids is rarely found in archaeal polar lipids. The total polar lipids extracted from archaeal species are negatively charged due to the preponderance of negatively charged polar head groups (Sprott, 1992). The total polar lipids extracted from some archaeal species such as Halobacterium salinarum consist entirely of archaeol type of core lipids, others such as from Methanobrevibacter smithii consist of a mixture of archaeol and caldarchaeol, whereas those from others such as Thermoplasma acidophilum consist predominantly of caldarchaeol cores (reviewed in Patel and Sprott, 1999).
The total lipid extract (contains neutral and the polar lipids), total polar lipid extract or purified polar lipids from archaeal species can be made into liposomes, which are referred to as archaeosomes (reviewed in Patel and Sprott, 1999). Archaeosomes prepared from polar lipids consisting exclusively of archaeol type cores, form a bilayer vesicle membrane as observed with conventional liposomes. Those made from polar lipids consisting exclusively of caldarchaeol type cores form a monolayer membrane that spans the entire vesicle membrane. Those made from polar lipids consisting of mixtures of both archaeol and caldarchaeol type cores form a vesicle membrane consisting of both mono- and bilayer membrane (Patel and Sprott, 1999).
In earlier studies, it was reported that archaeosomes elicit antigen-specific humoral antibody (Th2) and cell-mediated (Th1) immune responses, including CD8+ cytotoxic T lymphocyte (CTL) responses, to entrapped antigens upon immunization of the host by parenteral routes such as i.p., s.c. and i.m. (reviewed in Patel and Chen 2005; Sprott et al., 2000, 2001). Further, there has been no prior suggestion or indication that such archaeosome vaccines would be capable of eliciting mucosal immunity upon systemic or mucosal route of vaccine delivery. Prior art has indicated that adjuvants that help elicitation of systemic immune responses are generally incapable of eliciting mucosal responses upon administration of the vaccine by a mucosal route (Ryan et al., 2001; Singh and O'Hagan, 2002).
The citation of above references is not an admission that any of the foregoing is pertinent prior art. Representations as to the contents of these references and as to the dates of publication are based on the information available to the applicants and do not constitute any admissions as to correctness of the contents or the dates of the said references.